Recent progress made in the studies on piscine genes has developed various techniques for the extraction of genomic DNA from fish.
One of such techniques is to extract DNA from fish liver, fin or muscle, or even the whole body of small fish, which is based on the technique developed for mammals(see Brem, G. B., et al., Aquaculture, 68, 209-219(1988); Dunham, R. A., et al., Trans. Am. Fish Soc., 116, 87-91(1987); Ivics, Z., et al., Mol. Marine Biol. Biotechnol., 2, 162-173(1993); Khoo, H. W., et al., Aquaculture, 107, 1-19(1992); Penman, D. J., et al., Aquaculture, 85, 35-50(1990); Penman, D. J., et al., Mol. Repr. Dev., 30, 201-206(1991); and Zhang, P., et al., Mol. Repr. Dev., 25, 3-13(1990)). Another process developed for extracting DNA from salmonid fish is also based on the technique for mammals(see Taggart, J. B., et al., J. Fish Biol., 40, 963-965(1992)). However, these techniques are very complicated and require expensive proteinases, RNases, thermostatic apparatus, and a great deal of labor. Therefore, the analysis of large number of samples may be tiresome, laborious, and not economical in many situations. Further, fish is inevitably sacrificed or injured seriously in case of these methods.
A method for extracting genomic DNA from fish blood or sperm has been recently developed for the purpose of DNA fingerprinting(Cummings, S. A. and G. H. Thorgaard, Biotechniques, 17(3), 426-430(1994)). In this process, blood is obtained from the heart of an anesthetized salmon by using a syringe containing 1.times.SSC(0.15M NaCl, 0.015M sodium citrate) and the blood sample so obtained is put into a tube. A large amount of distilled water is added to the tube to disrupt the cells, and then, 5.times.SSC is added to make the solution isotonic. The mixture is centrifuged to obtain precipitate, which is washed with NaCl and EDTA, and then centrifuged again to obtain precipitate. A buffer solution containing proteinase K is added to the precipitate and the mixture is stirred at 60.degree. C. overnight to digest the precipitate. The mixture is cooled to a room temperature and ethanol is added to precipitate DNA, which is then separated and washed with ethanol. The obtained DNA is dried and dissolved in 1.times.TE buffer at 37.degree. C. overnight. In case of sperm, a buffer solution containing proteinase K is added and the mixture is maintained at 60.degree. C. overnight to digest the sperm; and then the same procedures as in the case of blood are repeated.
However, in this process, expensive proteinase K is employed for the extraction of DNA, which in turn requires a thermostatic apparatus and a stirrer. Further, it takes a long time to complete the process. More importantly, a considerable amount of proteins may precipitate together with DNA during the process.
Therefore, efforts have continued for the development of a simple and economical process for the extraction of genomic DNA from fish.